SERCA2a is a critical ATPase responsible for Ca2+ re-uptake by cardiac muscle cells during excitation-contraction coupling. The down-regulation of SERCA2a is one of the primary abnormalities found in failing hearts. Consistent with this observation, restoration of SERCA2a by gene transfer has proven to be effective in normalizing cardiac function in humans as well as model animals.
Heart failure (HF) represents complex patho-physiological conditions that are often final consequences of various cardiovascular disorders including atherosclerosis, cardiomyopathy, and hypertension. The incidence of HF continues to grow worldwide. HF is characterized by contractile dysfunction that is in large part due to abnormalities in sarcoplasmic reticulum (SR) Ca2+ cycling (Gwathmey et al., 1987; Gwathmey and Hajjar, 1990). In normal human cardiomyocytes, the activity of SERCA2a contributes to the removal of more than 70% of cytosolic Ca2+ into the SR during diastole. SERCA2a, therefore, affects muscle contraction kinetics by determining the SR Ca2+ content in the subsequent beat (MacLennan and Kranias, 2003). Impaired SR Ca2+ uptake due to a decreased expression level and a reduced activity of SERCA2a has been reported in failing human hearts (Meyer et al., 1995; Minamisawa et al., 1999; Zarain-Herzberg et al., 1996). It is known that restoration of SERCA2a levels by gene transfer improves systolic and diastolic dysfunction in rodent (del Monte et al., 2001) and porcine models of HF (Byrne et al., 2008; Kawase et al., 2008).
Post-translational modification (PTM) is an important way to modulate the function of diverse cellular proteins by affecting their enzymatic activity, localization, stability, or turnover rates in response to environmental stimuli. It was previously shown that SERCA2a activity could be modulated by PTM such as glutathiolation (Adachi et al., 2004; Dremina et al., 2007; Lancel et al., 2009) and nitration (Knyushko et al., 2005). It is also known that the isoelectric point of SERCA2a becomes both more acidic and basic in the failing heart compared to the normal heart. In addition, restoration of SERCA2a levels by gene transfer also partially restored this shifted isoelectric point of SERCA2a in the failing heart (Figure S1). These data indicated that there existed multiple PTMs of SERCA2a, which are associated with the development of HF.
Small ubiquitin-related modifier (SUMO), which shares 18% sequence homology with ubiquitin, can be conjugated to lysine residues of target proteins. This PTM is referred to as SUMOylation. In humans, three SUMO isoforms (SUMO1-3) appear to modify both common and distinct substrates (Welchman et al., 2005). Specifically, SUMO1 has been shown to play important roles in modulating diverse cellular processes including transcriptional regulation, nuclear transport, DNA repair, cell cycle, plasma membrane depolarization, and signal transduction both in normal and pathogenic conditions (Sarge and Park-Sarge, 2009). SUMO-mediated regulation of cardiac transcriptional factors such as GATA4 (Wang et al., 2004) and Nkx2.5 (Wang et al., 2008) is associated with differentiation of cardiomyocytes and development of cardiac structures. SUMO-mediated modification also regulates cardiac ion channel activity including voltage-gated potassium channels (Benson et al., 2007). In addition, SUMOylation of ERK5 has recently been linked to diabetes-related heart conditions (Shishido et al., 2008).
Over the past few years, a host of studies has shown that SUMOylation can regulate the activities of a variety of proteins both in normal and human pathogenesis, including neurodegenerative diseases, cancer, and familial dilated cardiomyopathy (Kim and Baek, 2006; Steffan et al., 2004; Zhang and Sarge, 2008). Recently, the critical role of SUMOylation of ERK5 in diabetic heart has been reported (Woo and Abe, 2010).
SUMOylation can affect biochemical properties of target proteins such as enzymatic activities and stabilities. The underlying molecular mechanism is largely unknown, but three possibilities have been suggested. SUMO attachment may alter interaction between the target and its binding partners (DNA or protein) by masking of existing binding sites or addition of interfaces that are present in SUMO. Alternatively, SUMOylation may induce a conformational change of the target proteins, which can either increase or decrease the enzymatic activities. Finally, SUMOylation can block other PTMs at lysine residues such as ubiquitination and acetylation, which lead to alterations in the functional properties of target proteins.
It has been shown that SERCA2a is a target of oxidative PTMs. Accumulated nitration of SERCA2a has been observed in skeletal muscle undergoing electrical stimulation, in hypercholesteremic aorta, and in ischemic human heart. The nitration at tyrosines 294 and 295 was correlated with the reduced Ca2+− ATPase activity of SERCA2a. The position of these tyrosines within a functionally key membrane region of SERCA2a and close to a negatively charged side chain would seem to ensure both efficient nitration and a mechanism for decreased rates of calcium transport. In addition, oxidation of a redox-sensitive cysteine residue of SERCA2a (cysteine 674) was detected in diabetic pigs (Ying et al., 2008). This oxidative modification may be related to the accelerated SERCA degradation in ischemic heart (French et al., 2006) and in H9c2 cells exposed to hydrogen peroxide Mara et al., 2005).
Accordingly, a need continues to exist in the art for therapeutics and methods of treating cardiovascular disease such as heart failure in a manner that is safe and effective for humans and other animals.